Optimization of the Separation Method for Bioleaching Bacteria DNA and RNA Extracted Simultaneously

Abstract

DNA and RNA based analysis was a useful way to characterize microbial communities during biohydrometallurgical processes. However, feasible, affordable and efficient DNA and RNA separation methods are rarely reported although several simultaneous DNA and RNA extraction methods have been developed recently. In this study, various salts including NaCl, CaCl2 and LiCl were tested for separation of DNA and RNA. Salt concentration, nucleic acid concentration, precipitation temperature and time were optimized. The results showed that LiCl was more efficient to separate DNA and RNA than the other two salts. The optimized conditions were as follows: 1/4 volume saturated LiCl was used for precipitating RNA first at -20°C for 30 min for total nucleic acid concentration of approximately 200-400 ng/μL, and then centrifuged at 12,000×g at room temperature for 20 min to collect RNA. DNA in the supernatant was precipitated using 0.6 volume isopropanol and then collected by centrifugation at 12,000×g at room temperature for 20 min. The results indicated that DNA and RNA could be extracted from not only pure culture of Acidithiobacillus ferrooxidans(A.ferrooxidans), Acidithiobacillus caldus(A. caldus), Acidithiobacillus albertensis(A. albertensis), Leptospirillum ferrooxidans(L. ferrooxidans), Ferroplasma thermophilium(F. thermophilium), but also the acid mine drainage(AMD) water samples from Daye copper mine, Xiangxi gold mine, Axi gold mine. The resulted DNA and RNA could be amplified and A260/280 was 1.8-2.0, A260/230 was 1.8-2.2, which indicated high quality of DNA and RNA. This method could be widely used for separation of bioleaching bacteria DNA and RNA extracted simultaneously.